EDI™ Quantitative SARS-CoV-2 Neutralizing Antibody ELISA Kit

Enzyme Linked Immunosorbent Assay (ELISA) for the quantitative detection of the SARS-CoV-2 neutralizing antibody concentration in a human serum.

REF KTR-1036 RUO 96

INTENDED USE
The EDI™ quantitative SARS-CoV-2 neutralizing antibody ELISA Kit is
2. Biotinylated ACE2 (31278)
an Enzyme-Linked Immunosorbent Assay (ELISA) kit intended for the
quantitative measurement of the neutralizing antibody to SARS-CoV-2 Biotinylated recombinant ACE2 protein.
receptor binding domain (RBD) of its spike protein in human serum. Qty: 1 x 5 mL
Testing is limited to laboratories certified under the Clinical Laboratory Storage: 2 – 8°C
Improvement Amendments of 1988 (CLIA), 42 U.S.C. 263a, to perform Preparation: Ready to use
moderate or high complexity tests. IgG antibodies to SARS-CoV-2 are
generally detectable in blood several days after initial infection, as well 3. HRP labeled Spike Protein (31279)
as in the convalescent stage. Laboratories within the United States and HRP labeled spike protein in a stabilized protein matrix.
its territories are required to report all positive results to the appropriate Qty: 1 x 5 mL
public health authorities. The EDI™ quantitative SARS-CoV-2 2 – 8°C
neutralizing antibody ELISA Kit may shows false higher antibody
Storage:
concentration due to cross-reactivity from pre-existing antibody or Preparation: Ready to use
other possible causes.
4. ELISA Wash Concentrate (10010)
For Research Use Only. Not for use in Diagnostic Procedures. Surfactant in a phosphate buffered saline with non-azide
reservative.
SUMMARY OF PHYSIOLOGY Qty: 1 x 30 mL
2019 novel coronavirus (2019-nCoV or SARS-CoV-2 or COVID-19) is Storage: 2 – 25°C
a single-stranded RNA coronavirus2. Comparisons of the genetic Preparation: 30x Concentrate. The contents must be diluted
sequences of this virus have shown similarities to SARS-CoV and bat with 870 mL distilled water and mixed well before
coronaviruses7. In humans, coronaviruses cause respiratory use
infections3. Coronaviruses are composed of several proteins including
the spike (S), envelope (E), membrane (M), and nucleocapsid (N) 4. 5. ELISA HRP Substrate (10020)
Results suggest that the spike protein retains sufficient affinity to the Tetramethylbenzidine (TMB) with stabilized hydrogen peroxide.
Angiotensin Converting Enzyme-2 receptor to use it as a mechanism Qty: 1 x 15 mL
of cell entry6. Human to human transmission of coronaviruses is 2 – 8°C
Storage:
primarily thought to occur among close contacts via respiratory
droplets generated by sneezing and coughing1. IgG is the most Preparation: Ready to use
abundantly found immunoglobulin to be produced in response to an
antigen and will be maintained in the body after initial exposure for long 6. ELISA Stop Solution (10030)
term response5. 0.5 M sulfuric acid.
Qty: 1 x 15 mL
ASSAY PRINCIPLE Storage: 2 – 25°C
This ELISA kit is designed, developed, and produced for the Preparation: Ready to use
quantitative measurement of the neutralizing antibodies to SARS-CoV-
2 RBD of its spike protein in human serum This assay utilizes the 7. nCoV Neutralizing Antibody Calibrator Level 1 (31281)
microplate-based enzyme immunoassay technique. A ready-to-use sample dilution buffer.
Assay calibrators, controls, and human serum samples are added to Qty: 1 x 15 mL
the microtiter wells of a microplate coated with streptavidin. Storage: 2 – 8°C
Simultaneously, horseradish peroxidase (HRP) labeled COVID-19 Preparation: Ready to use
recombinant spike protein and biotinylated angiotensin converting
enzyme-2 (ACE-2) are added to each well. After the first incubation 8. nCoV Neutralizing Antibody Calibrator Level 5 (31285)
period, the unbound protein matrix is removed with a subsequent Calibrators with a bovine serum albumin based matrix with non-
washing step; a complex of "Streptavidin---Biotin-ACE2---HRP-COVID- azide preservative. Refer to vials for exact concentration.
19 recombinant spike protein" is formed. If there is specific COVID-19 Qty: 1 x 0.5 mL
neutralizing antibody present in the tested specimen, the formation of Storage: 2 – 8°C.
the above complex is blocked. A color reaction with a substrate Preparation: Lyophilized powder (see Assay Procedure section)
solution in a timed reaction is measured in a spectrophotometric
microplate reader. The HRP enzymatic activity of the complex on the 9. nCoV Neutralizing Antibody Controls (31286 – 31287)
wall of the microtiter well is inversely proportional to the amount of the Controls with a bovine serum albumin-based matrix with non-azide
COVID-19 neutralizing antibody level in the tested specimen. preservative.
Qty: 2 x 0.5 mL
REAGENTS: PREPARATION AND STORAGE Storage: 2 – 8°C.
The test kit must be stored at 2 – 8°C. Refer the label on a kit box for
the expiration date. All components are stable until expiration date. Preparation: Lyophilized powder (see Assay Procedure section)

1. Streptavidin Coated Microplate (10040) SAFETY PRECAUTIONS

Microplate coated with streptavidin. The reagents are for in-vitro diagnostic use only. Source material
Qty: 1 x 96 well microplate which contains reagents of bovine serum albumin was derived in the
Storage: 2 – 8°C contiguous 48 United States. It was obtained only from healthy donor
Preparation: Ready to use animals maintained under veterinary supervision and found free of
KT-1036/IFU/RUO/V1/01-2021 Page |1
 EDI™ Quantitative SARS-CoV-2 Neutralizing Antibody ELISA Kit
Enzyme Linked Immunosorbent Assay (ELISA) for the quantitative detection of the SARS-CoV-2 neutralizing antibody concentration in a human serum.

REF KTR-1036 RUO 96

contagious diseases. Wear gloves while performing this assay and Row Strip 1 Strip 2 Strip 3
handle these reagents as if they were potentially infectious. Avoid A Calibrator level 1 Calibrator level 5 Sample 2
contact with reagents containing hydrogen peroxide, or sulfuric acid. B Calibrator level 1 Calibrator level 5 Sample 2
C Calibrator level 2 Control 1 Sample 3
Keep out of reach skin, eyes and/or clothing. Do not ingest or inhale D Calibrator level 2 Control 1 Sample 3
fumes. On contact, flush with copious amounts of water for at least 15 E Calibrator level 3 Control 2 Sample 4
minutes. Exercise Good Laboratory Practices. F Calibrator level 3 Control 2 Sample 4
G Calibrator level 4 Sample 1 Sample 5
H Calibrator level 4 Sample 1 Sample 5
MATERIALS REQUIRED BUT NOT PROVIDED 3. Add 25µL of calibrators, controls, and unknownsamples into
1. Precision single channel pipettes capable of delivering 10 µL,
the designated microwells.
20 µL, 100 µL, and 1000 µL, etc.
2. Repeating dispenser suitable for delivering 100 µL. 4. Add 50 µLof HRP labeled spike protein (31279) into each
3. Disposable pipette tips suitable for above volume dispensing. microwell.
4. Disposable 12 x 75 mm or 13 x 100 mm glass or plastic tubes. 5. Add 50 µLof biotinylated ACE2 (31278) into each microwell.
5. Disposable plastic 1000 mL bottle with caps. 6. Mix gently and cover the plate with one plate sealer and
6. Aluminum foil. aluminum foil. Incubate at room temperature (20-
7. Deionized or distilled water. 25ºC)for45 minutes.
8. Plastic microtiter well cover or polyethylene film.
7. Remove the plate sealer. Aspirate the contents of each well.
9. ELISA multichannel wash bottle or automatic (semi-automatic)
washing system. Wash each well 5 times by dispensing 350 µL of diluted
10. Spectrophotometric microplate reader capable of reading wash solution (10010) into each well, and then completely
absorbance at 450 nm. aspirate the contents. Alternatively, an automated microplate
11. Calibrated Timer. washer can be used.
8. Add 100 µL of the substrate (10020) into the microwells.
SAMPLE COLLECTION & STORAGE 9. Mix gently and cover the plate with aluminum foil. Incubate
Only 50 μL of human serum is required for measurement in duplicate. at room temperature (20-25 ºC) for 20 minutes.
Samples should only be used on the same day or stored below -20oC. 10. Remove the aluminum foil and add 100 µL of stop solution
Severe hemolytic samples should not be used. (10030) into each of the microwells. Mix by gently by tapping
the plate.
ASSAY PROCEDURE 11. Read the absorbance at 450 nm within 10 minutes with a
1. Reagent Preparation microplate reader.
1. Prior to use, allow all reagents to come to room temperature.
Reagents from different kit lot numbers should not be
combined or interchanged. PROCEDURAL NOTES
2. ELISA Wash Concentrate (10010) must be diluted to 1. Calibrator L4 can be made by mixing 200 µL of calibrator L5 with
working solution prior to use. Please see REAGENTS 400 µL of calibrator 1. Calibrator L3 can be made by mixing 200
section for details. µL of calibrator L4 with 400 µL of calibrator 1. Calibrator L2 can
3. Reconstitute nCoV Neutralizing Antibody Calibrator Level 5 be made by mixing 200 µL of calibrator L3 with 400 µL of
(31285) by adding 0.5 mL deionized water. calibrator 1.
4. Prepare calibrator level 2, 3 and 4by 1:3 serial dilutions of 2. It is recommended that all samples be assayed in duplicate. The
level 5 (31285) with nCoV Neutralizing Antibody Calibrator average absorbance reading of each duplicate should be used for
Level 1 (31281). Assay calibrators should be used within 2 data reduction and the calculation of results.
hours and should be stored below -20°C. Do not exceed 3 3. Keep light-sensitive reagents in the original bottles and avoid
freeze-thaw cycles. The calibrator concentrations are unnecessary exposure to the light.
indicated in the certificate of analysis of the kit. 4. Store any unused antibody-coated strips in the foil ziploc bag with
desiccant to protect from moisture.
5. Careful technique and use of properly calibrated pipetting devices
are necessary to ensure reproducibility of the test.
6. Incubation time(s)and/or temperature(s) other than those stated in
the package insert may affect the results.
7. Avoid air bubbles in the microwell as it could result in lower
binding efficiency and higher CV% of duplicate reading.
8. All reagents should be mixed thoroughly and gently prior to use.
Avoid foaming.

QUALITY CONTROL
To assure the validity of the results each assay should include
adequate controls with known SARA-CoV-2 neutralizing
2. Assay Procedure
antibody levels. EDI recommends including own laboratory
1. Place a sufficient number of microwell strips (10040) in a controls in addition to those provided with thekit.
holder to run the calibrators, controls, and samples in
duplicate.
2. Test Configuration
KT-1036/IFU/RUO/V1/01-2021 Page |2
 EDI™ Quantitative SARS-CoV-2 Neutralizing Antibody ELISA Kit
Enzyme Linked Immunosorbent Assay (ELISA) for the quantitative detection of the SARS-CoV-2 neutralizing antibody concentration in a human serum.

REF KTR-1036 RUO 96

INTERPRETION OF RESULTS
1. Calculate the average absorbance for each pair of duplicate test
results.
2. The calibration curve is generated by the absorbance of all
calibrator levels on the ordinate against the calibrator
concentration on appropriate computer assisted data reduction
programfor the calculation of results.
3. It is recommended to use following curve fits: (1) 4-Parameteror
(2) Point-to-Point.
4. The SARS-CoV-2 neutralizing antibody concentrations for the
controls and patient samples are read directly from the calibration
curve using their respective absorbance.

LIMITATIONS OF THE PROCEDURE

1. The values of the assay calibrators were established by diluting
ahuman SARS-CoV-2 neutralizing antibodystock in a phosphate
buffer protein matrix.
2. Patients with low immunity or other diseases that affect immune EXPECTED VALUES
function, failure of critical systemic organs, and use of drugs that One hundred donor serum samples from December 2018 to February
suppress immune function can also lead to negative results. 2019 were collected and tested. The average concentration of SARS-
Previous infection of SARS or other coronavirus strains may CoV-2 neutralizing antibody was found to be 1.13 U/mL with a median
present a low-level SARS-CoV-2 neutralizing antibodydue to at 0.64 U/mL and standard deviation at 2.91 U/mL. The manufacturer
similarity of different strains. recommended P97.5 cut-off level is 5 U/mL for the present of
3. Water deionized with polyester resins may inactive the
neutralizing antibody in test sample. It is highly recommended that
horseradish peroxidase enzyme.
4. Laboratories within the United States and its territories are each laboratory should establish their own cut off level based on local
required to report all positive results to the appropriate public population.
health authorities.
5. The EDI™ quantitative SARS-CoV-2neutralizing antibody ELISA
Kit is only for use under the Food and Drug Administration’s WARRANTY
Emergency Use Authorization. This product is warranted to perform as described in its labeling and
literature when used in accordance with all instructions. Epitope
EXAMPLE DATA (Calibration Curve) Diagnostics, Inc. DISCLAIMS ANY IMPLIED WARRANTY OF
This ELISA calculates the concentration values IgG of the samples and MERCHANTABILITY OR FITNESS FOR A PARTICULAR PURPOSE,
the controls by a calibration curve (fitting method: four parameters or and in no event shall Epitope Diagnostics, Inc. be liable for
point-to-point) and the measured absorbance.The following is a typical consequential damages. Replacement of the product or refund of the
calibration curve: purchase price is the exclusive remedy for the purchaser. This
warranty gives you specific legal rights and you may have other rights,
which vary from state to state.
Well ID OD 450 nm Average

Calibrator Level 1: 2.012

0 U/mL
2.047 REFERENCES
2.082
1. CDC (2020). Transmission of Novel Coronavirus (COVID-19).
Calibrator Level 2: 1.203 2. Chenjia Yuan, Shi Jinsong, Qiudong An, Liu Chang, Li Xin, Qiang,
1.228
3.7 U/mL 1.252 Ruanji Shou, mountains. Wuhan 2019 Bioinformatics coronavirus
Calibrator Level 3: 0.586 genome analysis [J / OL]. Bioinformatics: 1-10 [2020-02-10].
0.570
11.1 U/mL 0.555 3. Fehr, A. R., & Perlman, S. (2015). Coronaviruses: An Overview of
Calibrator Level 4: 0.263
0.263 Their Replication and Pathogenesis. Coronaviruses Methods in
33.5 U/mL 0.264 Molecular Biology, 1–23. doi: 10.1007/978-1-4939-2438-7_1
Calibrator Level 5:
0.196 4. Li, F., Li, W., Farzan, M., & Harrison, S. (2005). Structure of SARS
0.195 0.196 coronavirus spike receptor-binding domain complexed with its
100 U/mL
0.998 receptor. doi: 10.2210/pdb2ajf/pdb
5. Wu, L.-P., Wang, N.-C., Chang, Y.-H., Tian, X.-Y., Na, D.-Y., Zhang,
Note: This curve should not be used in lieu of calibrator curve run
L.-Y., Liang, G.-D. (2007). Duration of Antibody Responses after
with each assay.
Severe Acute Respiratory Syndrome. Emerging Infectious Diseases,
13(10), 1562–1564. doi: 10.3201/eid1310.070576
6. Xu, X., Chen, P., Wang, J., Feng, J., Zhou, H., Li, X., Hao, P.
(2020). Evolution of the novel coronavirus from the ongoing Wuhan
outbreak and modeling of its spike protein for risk of human
transmission. Science China Life Sciences. doi: 10.1007/s11427-
020-1637-5
7. Zhou, P., Yang, X.-L., Wang, X.-G., Hu, B., Zhang, L., Zhang, W.,
Shi, Z.-L. (2020). A pneumonia outbreak associated with a new
coronavirus of probable bat origin. Nature. doi: 10.1038/s41586-020-
2012-7
KT-1036/IFU/RUO/V1/01-2021 Page |3
 EDI™ Quantitative SARS-CoV-2 Neutralizing Antibody ELISA Kit
Enzyme Linked Immunosorbent Assay (ELISA) for the quantitative detection of the SARS-CoV-2 neutralizing antibody concentration in a human serum.

REF KTR-1036 RUO 96

TECHNICAL ASSISTANCE AND CUSTOMER SERVICE

For technical assistance or place an order, please contact Epitope
Diagnostics, Inc. at +1 (858) 693-7877,fax:+1 (858) 693-7678 or email:
[email protected]

This product is manufactured by

Epitope Diagnostics, Inc.
7110 Carroll Road
San Diego, CA 92121, USA

Please visit our website at www.epitopediagnostics.com to learn more

about our products and services.

GLOSSARY OF SYMBOLS (EN 980/ISO 15223)

RUO
IVD LOT
For Research Use
In Vitro Diagnostic Lot Number
Only
Device

REF
Catalog Number Read instructions Number of Tests
before use

Keep away from

Store at Use by heat and direct sun
light

Manufacturer

KT-1036/IFU/RUO/V1/01-2021 Page |4